Journal of Crop Improvement
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/wcim20
Cross-pollination effects on morphological,
molecular, and biochemical diversity of a selected
cinnamon (Cinnamomum zeylanicum Blume)
seedling population
N. M. N Liyanage , A. L. Ranawake & P. C. G. Bandaranayake
To cite this article: N. M. N Liyanage , A. L. Ranawake & P. C. G. Bandaranayake (2020): Crosspollination effects on morphological, molecular, and biochemical diversity of a selected cinnamon
(Cinnamomum�zeylanicum Blume) seedling population, Journal of Crop Improvement, DOI:
10.1080/15427528.2020.1795769
To link to this article: https://doi.org/10.1080/15427528.2020.1795769
Published online: 21 Aug 2020.
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JOURNAL OF CROP IMPROVEMENT
https://doi.org/10.1080/15427528.2020.1795769
Cross-pollination effects on morphological, molecular,
and biochemical diversity of a selected cinnamon
(Cinnamomum zeylanicum Blume) seedling population
N. M. N Liyanage
a,b
, A. L. Ranawakec, and P. C. G. Bandaranayake
a
a
Agricultural Biotechnology Centre, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri
Lanka; bPostgraduate Institute of Science, University of Peradeniya, Peradeniya, Sri Lanka; cDepartment
of Agricultural Biology, Faculty of Agriculture, University of Ruhuna, Matara, Sri Lanka
ABSTRACT
Cinnamomum zeylanicum Blume, also known as true cinnamon,
has gained more attention recently because of its proven
medicinal properties. Having uniform quality raw materials is
the key to the sustainability of pharmaceutical and other
industries. Nevertheless, the majority of the cinnamon planta­
tions in Sri Lanka and elsewhere have originated from highly
cross-pollinated seeds. However, no reported studies exist on
the variability of individuals caused by the natural crosspollination of cinnamon. Therefore, our work focused on mor­
phological, genetic, and biochemical characterization of
a population of individuals that resulted from a single crosspollination event of a known C. zeylanicum mother plant. The
mother plant, the possible pollen donor and the selected
progeny were assessed with several age- and environmentindependent morphological traits and several Inter Simple
Sequence Repeat (ISSR) regions. Progeny had significantly dif­
ferent leaf and apex shapes and ISSR regions were about 80%
polymorphic compared to the parents. The morphological and
genetic diversity of progeny was also represented in the cin­
namaldehyde, cinnamyl alcohol and cinnamyl acetate content
at the seedling stage.
ARTICLE HISTORY
Received 20 April 2020
Accepted 7 July 2020
KEYWORDS
True cinnamon; Ceylon
cinnamon; cinnamon
plantation; cinnamon
propagation; ISSR
Introduction
Cinnamomum zeylanicum Blume (1826) (Cinnamon) is native to Sri Lanka.
It has gained more attention during recent years because of a vast amount of
scientific evidence on its potential medicinal and therapeutic values (Zare
et al. 2019; Sadeghi et al. 2019). Sri Lanka is the largest producer of true
cinnamon or the Ceylon cinnamon in the world, accounting for about
65–70% of the global production (MDSIT and SLEDB 2018).
Cinnamon is an evergreen perennial that belongs to the family Lauraceae in
the genus Cinnamomum, which comprises about 340 species of trees and
CONTACT P. C. G. Bandaranayake
pradeepag@agri.pdn.ac.lk; pgunathilake@ucdavis.edu
Agricultural
Biotechnology Centre, Faculty of Agriculture, University of Peradeniya, 20400, Peradeniya, Sri Lanka.
Supplementary data for this article can be accessed here.
© 2020 Informa UK Limited, trading as Taylor & Francis Group
2
N. M. N. LIYANAGE ET AL.
shrubs (“The Plant List” 2013). Besides true cinnamon, Cinnamomum cassia
Presl (Chinese cassia), Cinnamomum tamala Nees (Indian cassia),
Cinnamomum aromaticum Nees (Chinese cinnamon), Cinnamomum urman­
nii Nees (Indonesian cassia), Cinnamomum loureirii Nees (Saigon cassia) and
Cinnamomum camphora (Camphor) are also economically important
(Krishnamoorthy et al. 1997). In addition to C. zeylanicum, the following
seven endemic wild relatives of cinnamon in Sri Lanka are distributed in
different agro-ecological zones: Cinnamomum citriodorum (Pengiri kurundu),
Cinnamomum capparu-coronde (Kapuru kurundu), Cinnamomum dubium
(Sevel kurundu), Cinnamomum litseaefolium (Kudu kurundu), Cinnamomum
ovalifolium (Wal kurundu), Cinnamomum rivulorum and Cinnamomum sin­
harajaense (Kumarathilake et al. 2010; Wijesinghe and Pathirana 2000).
Cinnamon has gained much attention in the past decade or so, mainly
because of its medicinal properties, viz., anti-microbial, anti-parasitic, antioxidant, anti-inflammatory, anti-diabetic, anti-ulcer, cardiovascular diseaselowering properties (Ravindran, Babu, and Shylaja 2004; Vangalapati et al.
2012; Rao and Gan 2014; Nabavi et al. 2015) and lipid-lowering abilities
(Vangalapati et al. 2012). Such properties are attributed to unique secondary
metabolic profiles in both bark and leaf of C. zeylanicum, including cinna­
maldehyde, eugenol, cinnamyl alcohol, cinnamyl acetate, coumarin, and
coumaric acid (Zachariah and Leela 2018).
Metabolic profile of plants heavily depends on the genetic basis, physio­
logical status, such as age and maturity of the plant, and the macro- and
micro-environmental conditions that the plant is exposed to (Motta et al.
2019; Jaakola and Hohtola 2010; Sampaio, Edrada-Ebel, and Costa 2016;
Schwachtje et al. 2019). Therefore, it is essential to minimize such variations
to maintain the quality of the produce. This is even more critical when the
harvest is used in nutraceutical and therapeutic industries.
Since the genetic basis is critical in deciding the quality and the quantity of
final yield, additional efforts are needed for selecting superior genotypes and
maintaining genetic uniformity. As a step forward for further improving the
quality of Ceylon cinnamon, the Department of Export Agriculture (DEA),
Sri Lanka, has released two varieties, Sri gamunu (SG) and Sri wijaya (SW)
based on long-term selection (Wijesinghe and Pathirana 2000). Nevertheless,
the benefits do not reach farmers since cinnamon is mainly propagated
through seed. Current vegetative propagation methods are not sufficiently
efficient to satisfy the planting material demand. Further, vegetatively pro­
pagated materials are not popular among farmers because of some unsolved
crop management and maintenance issues.
Interestingly, the biology of C. zeylanicum flower and flowering pattern
promote genetic diversity among its offspring. C. zeylanicum has a complete
flower but with an unusual behavior known as protogynous dichogamy
(Rohwer 1993), where female and male flowers are separated temporally,
JOURNAL OF CROP IMPROVEMENT
3
promoting cross-pollination. Therefore, planting genetically diverse crosspollinated seeds affect the quality and quantity of varieties.
Morphological and biochemical diversity of C. zeylanicum seedling pro­
genies has previously been studied; however, no parental lines or genetic
analyzes were included (Paul and Sahoo 1993). Abeysinghe et al. (2009)
studied species-level diversity using TrnL intron regions, intergenic spacers
between trnT-trnL, trnL-trnF, trnH- psbA and nuclear internal transcribed
spacer (ITS). Abeysinghe and colleagues also studied species-level diversity of
some Sri Lankan cinnamon accessions using randomly amplified poly­
morphic DNA (RAPD) and sequence-related amplified polymorphic
(SRAP) markers (Abeysinghe et al. 2014). However, within-species diversity
has not been sufficiently considered.
Since continuous seed propagation has been practiced for centuries, exist­
ing commercial plantations and germplasm collections are expected to be
morphologically and genetically heterogeneous. To estimate such genetic
variations, a seedling population consisted of 10 individuals collected from
a selected mother plant, which resulted from a cross-pollination event, was
assessed. The Inter Simple Sequence Repeat (ISSR) regions were selected for
molecular analysis, and the same seedlings were assessed for the morpholo­
gical and biochemical diversity under controlled environmental conditions.
Materials and methods
Sample collection
The DEA maintains a vegetatively propagated plantation of SW and SG
varieties grown in alternate rows, in the Mid country Wet zone (WM2b) of
Sri Lanka at GPS coordinates of 7°11ʹ57.0”N and 80°37ʹ39.3”E. Twenty-five
ripened seeds were harvested from an identified SW mother plant from the
above plantation and planted under controlled environmental conditions at
Mid country Wet zone at GPS coordinated of 7°15ʹ06.4”N and 80°35ʹ37.6”E.
Of them, 17 seeds were germinated, and 10 were selected randomly for the
study. Leaf samples were harvested from the mother plant, the adjacent SG
plant, and the oldest known cinnamon tree in the Royal Botanical Garden of
Sri Lanka (BG), planted in 1976.
Morphological characterization
Here we used the cinnamon descriptors developed by a team of scientists
from University of Ruhuna, Sri Lanka (TURIS 2013), for morphological
assessment. We considered the age- and environment-independent leaf
morphological characters, viz., leaf shape, leaf apex and leaf base. About
3–5 leaves from 10–12-week-old seedling and 10–15 young leaves from
4
N. M. N. LIYANAGE ET AL.
Figure 1. Leaf morphology of mother plants and seedlings. (a) 1. Sri wijaya mother plant 2. Sri
gamunu mother plant (scale bar 3 cm) (b) – (i) Sri wijaya seedlings (progeny of Sri wijaya mother
plant) W1, W2, W3, W4, W5, W8, W9 and W10 respectively (scale bar 3 cm). † Leaf shape: * oval,
** ovate-lanceolate, *** ovate, **** elliptic; Leaf apex: - → obtuse, ⇢ long acuminate, ⇞ acute, ⇨
acuminate;Leaf base: ► subcordate, ►► obtuse, ►►► rounded, ►►►► obtuse, contracted
into petiole, then shortly cuneate.
different branches of the mother plants were assessed and the scores were
averaged (Figure 1).
Genetic characterization
The genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit
(Qiagen 69106, Qiagen Sciences, Germantown, Maryland, USA) following
the manufacturer’s guidelines and DNA samples were stored at −20 ᴼC. The
quality and quantity of DNA samples were assessed with agarose gel electro­
phoresis and NanoDrop spectrophotometer (Nano2000, Thermo scientific,
Wilmington, Delaware, USA). Thirty-seven Inter Simple Sequence Repeats
(ISSR) regions from the list of University of British Colombia (UBC),
Canada, were assessed using SW, SG, and BG. Out of those, seven ISSR
primers that resulted in reproducible polymorphic bands were selected to
JOURNAL OF CROP IMPROVEMENT
5
assess the genetic diversity of the seedling population and mother plants.
Approximately similar amounts of DNA from each sample was amplified as
previously described by Pathirana et al. (2018) in 25 μL reaction volume
containing 5 μL of 5X Green Go Taq Buffer, 1.5 μL of 25 mM MgCl2, 0.4 μL
of 200 mM PCR nucleotide mix, 3 μL of 10 mM Spermidine, 2 μL of 10%
PVP, 1 μL of 10 mM primer, 0.1 μL of (1 U/μL) Go Taq flexi DNA
polymerase (Promega, Cat. No: M8295) and 10 μL of sterile distilled water
using a Verti Thermal Cycler (Applied Biosystems, USA). After initial dena­
turation at 94 ᴼC for 5 min, 30 cycles involving one-minute denaturation at
94 ᴼC, 30 seconds annealing at 47 ᴼC – 55 ᴼC, depending on primer
(Supplementary Table 1) one minute, extension at 72 ᴼC, followed by five
minutes final extension at 72 ᴼC, were completed. The polymerase chain
reaction (PCR) products were first resolved on 2% agarose gels buffered with
1X TAE, and the polyacrylamide gel electrophoresis (PAGE) was performed
with the same samples for an effective base pair separation for the identified
polymorphic primers. Eight percent acrylamide gels were run at 6 V/cm for
three hours in a vertical gel electrophoresis apparatus with 1X TBE buffer
and proceeded through staining and de-staining steps with 0.001 v/v of
ethidium bromide. The gels were visualized under a UV gel documentation
system (Chemi Doc TM XRS+ Molecular imager, Bio-Rad Laboratories Inc.,
Hercules, California, USA) and photographed.
Biochemical characterization
The commercial standards of trans-cinnamaldehyde, eugenol, coumarin,
coumaric acid, cinnamyl alcohol, and cinnamyl acetate, and all HPLCgrade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Immature stem samples of four randomly selected 15-week-old seedlings
were powdered using liquid nitrogen, and three technical replicates from
each plant were used for HPLC analysis.
To extract chemicals, 12 mL of 100% (v/v) methanol was added to 0.15 g
of each seedling-stem sample, sonicated for 20 min, and centrifuged at
7800 rpm for 10 min. The supernatant was then filtered through a 0.45 µm
nylon filter and used for HPLC analysis in an Agilent 1260 system equipped
with a Zorbax Eclipse plus C18, 5 µm, 4.6 mm × 150 mm column. Ten
microliters of each sample were injected and separated in a solvent gradient
between 0.1 (v/v) orthophosphoric acid and 100% (v/v) acetonitrile (ACN).
The ACN-time combinations were 80% (v/v) ACN for 0–20 min, 50% (v/v)
ACN for 20–30 min, and 100% (v/v) ACN for 30–45 min, at a flow rate of
1 mL per minute. Trans-cinnamaldehyde, eugenol, coumarin, coumaric acid,
cinnamyl alcohol, and cinnamyl acetate were identified with the retention
time of standards, and standard curves were generated with commercial
standards to assess the concentration.
6
N. M. N. LIYANAGE ET AL.
Data analysis
The experiment was arranged in a completely randomized design (CRD)
and the number of leaves per plant (3 to 6) was considered technical
replicates. Leaf morphological characters of mother plants and progeny
were analyzed using the Kruskal-Wallis test, and cluster analysis was con­
ducted using the Ward’s hierarchical algorithm based on Euclidean dis­
tances via IBM SPSS Statistics software (version 20), IBM, USA (SPSS Inc.,
Chicago, IL).
Well-separated and reproducible ISSR bands were scored using ‘1ʹ and ‘0ʹ
to indicate the presence and absence of bands, respectively, and a binary data
matrix was constructed. The number of total bands (TB), number of poly­
morphic bands (PB) and percentage of polymorphic bands (PPB) were
calculated for each primer. Marker performance was assessed from poly­
morphic information content (PIC), marker index (MI) and resolving power
(Rp). The PIC value for each dominant marker was calculated as:
PIC ¼ 1 ½f2þð1
f Þ2�;
where f is the frequency of amplified fragments and (1-f) is the frequency of
non-amplified fragments (Chesnokov and Artemyeva 2015). Average PIC
value from all loci per primer was used to calculate PIC value for each
primer. Effective multiplex ratio (EMR) was calculated according to
Chesnokov and Artemyeva (2015). The marker index for each primer was
obtained as a product of PIC and EMR (Kumar and Mishra 2014), as shown
below:
MI ¼ PIC � EMR
Further, the resolving power coefficient (Rp) was calculated for each poly­
morphic primer, according to Prevost and Wilkinson (1999). The binary data
were used for the inter-population analysis of mother plants and progeny
using POPGENE32 software (Yeh et al. 1997). The number of different alleles
(Na), number of effective alleles (Ne), number of polymorphic alleles (npl),
percentage polymorphic bands (ppb), Shannon information index (I) and
Nei’s genetic diversity (H) (Nei and Li 1979) were calculated.
Phylogenetic analysis was performed with the PHYLIP-3.695 phylogeny
inference package (Felsenstein 2002). The binary qualitative data matrix was
bootstrapped for 1000 resamples using seqboot program in PHYLIP-3.695
and subjected to restdist program to generate the fragment distance matrix
(Nei and Li 1979). Then the neighbor-joining program was used to con­
structs a tree by UPGMA (Unweighted Pair Group Method with Arithmetic
means) method and the consensus program was used to construct an
JOURNAL OF CROP IMPROVEMENT
7
unrooted tree with all groups that occurred more than 50% of the time and
viewed and annotated with Figtree Version 1.4.2 (Rambaut 2009).
Biochemical data were analyzed using analysis of variance (ANOVA) of
SAS 9.4 (SAS Institute Inc. Cary, North Carolina, USA). Statistical differences
between means were determined by the Duncan’s multiple range test at the
5% significance level.
Results
Age and environment-independent leaf morphological characters were used
for the morphological characterization. Of the leaf shapes, elliptic, narrowly
elliptic, ovate, oval and ovate-lanceolate types were prominent among both
SW and SG mother plants and progeny, whereas obtuse, acuminate, longacuminate, narrowly acuminate and acuminate with broad acumen leaf apex
types were shared in mother plants and progeny. Both SW and SG mother
plants and progeny had rounded, subcordate, obtuse and obtuse, contracted
into petiole, then shortly cuneate leaf base types (Figure 1). The SW and SG
mother plants showed significantly different leaf shapes and leaf apices
(Table 1). Similarly, those two characters were significantly different among
SW and SG mother plants and SW progeny as well. However, the leaf base
was not significantly different either between SW and SG or between SW or
SG mother plants and progeny.
Interestingly, the progeny seedlings consisted of several combinations of
leaf base-, shape- and apex-morphological characters, including parent types.
Further, several types of leaves could be seen within a single plant in the
progeny. Cluster analysis with morphology data (Figure 2) clustered SW and
SG together at a cluster distance of 6.25, showing more similarities among
considered leaf morphological characters, whereas four seedlings, W7, W8,
W9 and W10, grouped with parents at a cluster distance of 17.5. The other
plants (W5, W6, W1, W2, W3, and W4) clustered separately at a cluster
distance of 8.0.
The ISSR regions were used to assess the genetic diversity among
seedlings, and SG and SW mother plants. Out of 37 primers tested, only
22 primers resulted in clear, reproducible bands, with DNA from two SW
and SG mother plants and the oldest known C. zeylanicum live specimen
Table 1. Kruskal-Wallis test† results for leaf morphology characters.
Test combination
Sri wijaya mother plant and Sri gamunu mother plant
Sri wijaya mother plant, Sri gamunu mother plant
and 10 Sri wijaya seedlings
Leaf shape
0.015*
0.000*
Leaf apex
0.025*
0.000*
*Significant at α = 0.05.
†Kruskal-Wallis test conducted on the influence of three independent variables.
Leaf base
0.129
0.113
8
N. M. N. LIYANAGE ET AL.
Figure 2. Morphological differences of mother plants and seedlings. Cluster analysis using ward’s
hierarchical algorithm based on Euclidean distances for, leaf morphological characters; leaf
shape, leaf apex and leaf base by IBM SPSS Statistics software (version 20). P1: Sri gamunu
mother plant; P2: Sri wijaya mother plant; W1-W10: are the tested progeny (Sri wijaya seedlings)
of Sri wijaya mother plant.
in the Royal Botanical Garden. Only seven of them, i.e., UBC 808, UBC
834, UBC 835, UBC 840, UBC 841, UBC 842, and UBC 888, resulted in
polymorphic bands among three considered samples (data not shown).
Those primers amplified a total of 59 alleles, identified on 8% PAGE, with
an average of 8 bands per primer, of which an average of 4 bands was
polymorphic (Table 2). Since these primers provided sufficient
Table 2. Inter Simple Sequence Repeat (ISSR) primer parameters† obtained for mother plants
and oldest cinnamon sample.
Serial no.
1
2
3
4
5
6
7
Total
Primer code
UBC 808
UBC 834
UBC 835
UBC 840
UBC 841
UBC 842
UBC 888
Average/Primer
TB
14
8
3
4
6
10
14
59
8.429
PB
7
3
3
2
3
6
6
30
4.285
MB
7
5
0
2
3
4
8
29
4.142
PPB
50.0
37.5
100.0
50.0
50.0
60.0
42.9
390.4
50.85
PIC
0.222
0.167
0.444
0.222
0.222
0.267
0.190
1.735
0.250
Rp
1.143
1.111
1.111
1.000
0.889
1.111
0.889
7.254
1.030
EMR
3.500
1.125
3.000
1.000
1.500
3.600
2.571
15.254
2.328
MI
0.778
0.188
1.333
0.222
0.333
0.960
0.490
4.304
0.615
†TB = Total bands, PB = Polymorphic bands, PPB = Percentage of polymorphic bands, PIC = Polymorphic
information content, Rp = Resolving power of a primer, EMR = Effective multiplex ratio, MI = Marker index.
JOURNAL OF CROP IMPROVEMENT
9
Table 3. Inter Simple Sequence Repeat (ISSR) primer parameters† obtained for mother plants
and seedlings.
Serial no.
1
2
3
4
5
6
7
Total
Primer code
UBC 808
UBC 834
UBC 835
UBC 840
UBC 841
UBC 842
UBC 888
Average/Primer
TB
14
11
11
6
7
16
24
89
12.71
PB
11
10
10
5
5
9
22
72
10.28
MB
3
1
1
1
2
7
2
17
2.43
PPB
78.571
90.909
90.909
83.333
71.429
56.250
91.667
563.068
80.430
PIC
0.283
0.381
0.318
0.389
0.297
0.212
0.366
2.246
0.321
Rp
1.305
0.9333
0.8667
1.333
1.333
1.148
0.992
7.911
1.13
EMR
8.643
9.091
9.091
4.167
3.571
5.063
20.167
59.790
8.540
MI
2.443
3.464
2.891
1.621
1.061
1.073
7.381
19.930
2.840
†TB = Total bands, PB = Polymorphic bands, PPB = Percentage of polymorphic bands, PIC = Polymorphic
information content, Rp = Resolving power of a primer, EMR = Effective multiplex ratio, MI = Marker index.
polymorphism to differentiate SW and SG, the same set was used for
assessing seedlings in question. When the DNA from 10 seedlings, SG and
SW plants were used, a total of 89 alleles were resolved on 8% PAGE
(Table 3). Of them, 62 were polymorphic; the percentage of polymorph­
ism varied from 56% to 92%, with an average of 80%. The UBC 888
primer resulted in 22 polymorphic bands, whereas UBC 840 and UBC 841
produced only five polymorphic bands.
Generally, the performance of ISSR markers and their informativeness are
assessed with PIC, Rp, and EMR parameters (Kumar and Mishra 2014).
Similarly, the total utility of any marker system is estimated by MI, where
higher values indicate better systems (Chesnokov and Artemyeva 2015). MI
values among primers varied from a minimum of 1.061 in UBC 842 to
a maximum of 7.381 in UBC 888, with an average of 2.84 (Table 3).
Assuming Hardy-Weinberg equilibrium, Nei’s genetic diversity (H) was, on
average, 0.29 within SW progeny and mother plant, whereas Shannon’s
information index (I) was 0.44. The average number of different alleles
(Na) and the average number of effective alleles (Ne) were 1.80 and 1.50,
respectively (Table 4).
Cluster analysis based on Nei’s genetic distance is an accepted method to
assess the genetic similarity or dissimilarity among taxa. In the current
analysis, one of the seedlings, W6, separated from other seedlings and mother
plants at the rescaled distance of 250 (Figure 3). Within the large cluster,
some seedlings clustered together into small clusters, whereas W1 grouped
with the SW mother plant.
Table 4. Genetic diversity parameters† evaluated within mother plants and seedlings following
Nei’s method.
Population
Sample size PPB (%) npl
Na
Ne
H
I
Sri wijaya progeny with mother plants
12
80.90
72 1.8090 1.5043 0.2931 0.4363
†PPB = Percentage of polymorphic bands, npl = Number of polymorphic loci, Na = Number of different
alleles, Ne = Number of effective alleles, H = Nei’s diversity index, I = Shannon’s information index.
10
N. M. N. LIYANAGE ET AL.
Figure 3. Genetic relationship among mother plants and seedlings. Cluster analysis based on
genetic distance obtained from Inter Simple Sequence Repeat (ISSR) markers using Unweighted
Pair Group Method with Arithmetic means (UPGMA) method by PHYLIP 3.695 Software package.
P1: Sri gamunu mother plant; P2: Sri wijaya mother plant; W1-W10: are the tested progeny (Sri
wijaya seedlings) of Sri wijaya mother plant. Numbers on branches corresponds to bootstrap
values (1000 replications).
We were interested in knowing whether the observed morphological and
genetic variation contributed to the secondary metabolic composition of
individual plants. Four plants were selected for biochemical analysis, and
the trans-cinnamaldehyde, eugenol, coumarin, coumaric acid, cinnamyl alco­
hol and cinnamyl acetate in the sample were identified using the retention
times of the commercial standards measured at 290 nm, 285 nm, 280 nm,
290 nm, 265 nm, and 265 nm wavelengths, respectively. The concentration of
each compound was determined by developing a standard curve for each
component; R2 = 0.9979 for trans-cinnamaldehyde, R2 = 0.9999 for eugenol,
R2 = 0.9999 for coumarin, R2 = 0.9999 for coumaric acid, R2 = 0.9999 for
cinnamyl alcohol and R2 = 0.9999 for cinnamyl acetate. Interestingly, none of
the seedling-stem samples had eugenol, coumarin, and coumaric acid,
whereas trans-cinnamaldehyde, cinnamyl alcohol, and cinnamyl acetate var­
ied among seedling samples. The highest trans-cinnamaldehyde content,
3.424 mg/g, was detected in W8 seedling, whereas the lowest, 0.539 mg/g,
JOURNAL OF CROP IMPROVEMENT
11
Figure 4. Chemical composition of seedlings. (a) Mean concentration of chemical major chemical
compounds of Sri wijaya seedlings. Results are presented as average ± SE (in error bars) values of
three technical replicates. Representative (UV detection at 265 nm wavelength) HPLC elution
profiles of trans-cinnamaldehyde, cinnamyl alcohol and cinnamyl acetate in Sri wijaya seedlings
(b) W4 seedling (c) W7 seedling (d) W8 seedling (e) W10 seedling. One hundred and fifty
milligrams of shoot tissues were extracted in Absolute methanol, sonicated, and injected to
reverse phase HPLC gradient between ortho phosphoric 0.1% and acetonitrile 100. † W4, W7,
W8 and W10 are the randomly selected progeny (Sri wijaya seedlings) of Sri wijaya mother plant.
was found in W4 seedling. The average cinnamyl alcohol content of seedlings
varied from 0.084 mg/g in W8 to 0.224 mg/g in W4, whereas cinnamyl
acetate content varied from 0.039 mg/g in W8 to 0.639 mg/g in W4
(Figure 4). Interestingly, trans-cinnamaldehyde, cinnamyl alcohol, and cin­
namyl acetate contents were significantly different among the seedlings
(P < 0.05) (Table 5). Further, there were a few unidentified peaks in the
biochemical profiles, of which the presence and/or the highest peak varied
among the tested seedlings (Figure 4).
Discussion
While some attention has been given to morphological and biochemical
diversity of seedling populations of C. zeylanicum (Paranagama and
12
N. M. N. LIYANAGE ET AL.
Table 5. Chemical composition† of 15-week-old seedlings.
Cinnamon
seedling samples‡
W4
W7
W8
W10
Chemical compounds (mg/g)
Cinnamaldehyde
0.5390c ± 0.0337
0.9518c ± 0.0125
3.4236a ± 0.2552
1.6469b ± 0.0449
Cinnamyl alcohol
0.2238a ± 0.0016
0.0863c ± 0.0001
0.0842c ± 0.0007
0.2198b ± 0.0005
Cinnamyl acetate
0.6391a ± 0.0133
0.0437c ± 0.0001
0.0391c ± 0.0026
0.0858b ± 0.0002
†Values are means of three replicates (± SE). Means denoted by the same letter within a column are not
significantly different at P < 0.05.
‡W4, W7, W8 and W10 are the randomly selected progeny (Sri wijaya seedlings) of Sri wijaya mother plant.
Means denoted by the different letters (a, b & c) within a column indicate significant different at P < 0.05.
Mubarak 2001; Azad et al. 2018; Ford et al. 2019; Ding et al. 2011;
Jayaprakasha, Rao, and Sakariah 2002), only two reports exist on the mole­
cular analysis of C. zeylanicum in Sri Lanka (Abeysinghe et al. 2009, 2014).
Nevertheless, no previous attempts to assess the diversity of cinnamon were
made in a single cross-pollination event. Since the structure of the cinnamon
flower is such that it maximizes cross-pollination, such analysis is important
to plant breeding programs and to assess the uniformity of plantations.
Some plant traits are purely governed by genetics, whereas other pheno­
types are determined by a combination of genetics and environmental factors
(Motta et al. 2019; Sampaio, Edrada-Ebel, and Costa 2016). In the current
research, we compared the phenotypes of about four-year-old mother plants
growing in the field with two-month-old seedlings growing in a controlled,
protected house. Therefore, we could only use the age- and environmentindependent morphological traits proposed by Azard et al. (2015). Identified
leaf morphological characters of the offspring were compared with the
mother plant and a possible pollen donor to assess the variability created
by open pollination.
Our results suggested that the leaf shape and the leaf apex were the certain
age- and environment-independent leaf traits that could be used to compare
the selected progeny with mother plants. These traits can also be used for
screening progenies in breeding programs, as well. Paul and Sahoo (1993)
studied mature cinnamon seedling progenies of cinnamon in Orissa state,
India, using morphology and biochemical traits and reported wide variation
with respect to stem girth (7–16.6 cm), plant height (2.17–3.30 m), leaf oil
(0.38–1.80%), eugenol in leaf oil (traces to 80–98%) and bark oil. However,
they did not include possible parental lines, and their genetic diversity was
not assessed. Here we included the possible parents and morphological
diversity of seedlings suggested possible cross-pollination events among
adjacent plants, and the ISSR analysis further verified our observations.
ISSR regions are still widely used for assessing the genetic diversity of
species, especially when the genomic information is not available (Attanayake
et al. 2017; Alhasnawi, Mandal, and Jasim 2019). Four of the polymorphic
JOURNAL OF CROP IMPROVEMENT
13
primers used in the current work, UBC 834, UBC 841, UBC 840 and UBC
842, have previously been used in cinnamon genetic work, and several
investigators (Ho and Hung 2011; Uthairatsamee and Pipatwattanakul
2011; Dong et al. 2016) have reported high genetic variability
(H = 0.3–0.35, I = 0.4–0.5, Ne = 1.5–1.6) among the tested cinnamon
populations. When compared to previous work on cinnamon and other
cross-pollinated plants (Adhikari et al. 2015), our results suggest moderate
to high genetic diversity within SW progeny. For example, separate clustering
of W6 seedlings from the main cluster suggested that W6 was fairly different
from the others and the parent plants, whereas the rest of the seedlings
clustered with parents and others (Figure 3). While further information
with more primers is needed to resolve the genetic relationship of studied
individuals completely, available information suggests heterogeneity among
individuals created through cross-pollination.
Since all the seedlings were maintained under controlled environmental
conditions, variation in the biochemical composition could well be
a reflection of the genetic variation among the individuals. Interestingly,
W7 and W8 seedlings had comparable cinnamyl alcohol and cinnamyl
acetate contents, whereas W4 and W7 seedlings had similar cinnamaldehyde
content (Table 5). This is comparable to the genetic relationship where W4,
W7, and W8 clustered together within the main cluster (Figure 3).
Comparing complete metabolic profiles, especially at the harvest stage after
field planting, will provide further evidence for such relationships. Further,
the current study did not compare the biochemical profiles of the tested
seedlings with their mother plants because of the age differences.
Overall, our data suggest a considerably high level of cross-pollination in
C. zeylanicum. There are two types of plants in cinnamon, Type A and Type
B; identified according to the floral biology and flowering behavior
(Ravindran, Babu, and Shylaja 2004). While SG is considered a Type
A flower, the flower of SW is a Type B flower. Type A flower opens first as
a female flower on the first day, and the same flower will reopen the next day
as a male flower. There is a high possibility of type A flower of SG getting
pollinated with type B of SW when it opens the first time. Since SW and SG
are planted in alternate rows, cross-pollination is further promoted in the
selected plantation. Nevertheless, a recent study on the flowering behavior of
C. zeylanicum variety SG and SW showed that both male and female flowers
of the same tree are open for a short period, which could facilitate selfpollination (Hathurusinghe and Bandaranayake 2018). More research on the
same line would help in producing high-quality cinnamon seedlings, possibly
by isolation of mother plants.
Unfortunately, a majority of cinnamon plantations in the world are seed
propagated. Even newly introduced varieties bred for high quality and
quantity of yield, are currently propagated through seeds. Therefore, it is
14
N. M. N. LIYANAGE ET AL.
essential either to solve the field-management issues of vegetatively propa­
gated plants or to produce quality seeds through controlled pollination. The
development of molecular markers for early identification of quality planting
materials would be another option. Our results also provide evidence for the
possibility of utilizing ISSR markers for the identification of genotypes
similar to SW or SG. For example, the ISSR profile generated with UBC
842 primer is useful in separating SW and SG seedlings at an early stage.
Such selection would help in upgrading the quality of C. zeylanicum planta­
tions in the country and elsewhere.
Acknowledgments
We thank staff of the Department of Export Agriculture Especially, Mr. R.A.A.K. Ranawaka,
Assistant Director Research and the staff of the Agricultural Biotechnology Center, Faculty of
Agriculture, University of Peradeniya, for their support throughout.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was funded by the Ministry of Primary Industries and Social Empowerment, Sri
Lanka, through the National Science Foundation of Sri Lanka [Grant 2016/SP/CIN/01].
Availability of data and materials
The data generated during the current study are available from the corresponding author on
reasonable request.
ORCID
N. M. N Liyanage
http://orcid.org/0000-0001-6349-3859
P. C. G. Bandaranayake
http://orcid.org/0000-0001-8241-1775
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